The Tox21 Program is a multiagency collaborative effort among the Environmental Protection Agency (EPA), Food and Drug Administration (FDA), the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS), and NCATS to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Genomic Toxicology group has expanded our neurotoxicology work using a superior model for neurons, LUHMES cells, and human conditionally immortalized dopaminergic neurons. We have developed a novel method to determine toxicity mechanisms and pathways, the Toxmatrix (Tong et al. manuscript submitted). This method uses qHTS technologies to produce a dose-response curve for each toxicant and for each toxicant paired with 70 modulator chemicals with known targets. Interactions were demonstrated in which a modulator shifts the cytotoxic potency of a toxicant by protecting or sensitizing neuronal cells from a neurotoxicant. Each interaction identifies a toxicity pathway for the toxicant, according to the target of the modulator. The Toxmatrix has identified and confirmed toxicity pathways for 14 neurotoxicants. For example, neurotoxicants that cause Parkinsons disease or Parkinsonism shared several toxicity pathways identifying two characteristic vulnerabilities of dopaminergic neurons. Transcriptional profiling of 6 Parkinsons neurotoxicants has also identified a sensitive biomarker that is being exploited to make a qHTS assay to screen chemical libraries for these neurodegenerative toxicants. The Genomic Toxicology and Computational Toxicology groups have also advanced high-throughput gene expression technology. In 2017, we completed development of an automatic heuristic algorithm that identifies the genes that show a dose-dependent response to each treatment and quantifies the Point of Departure (POD), the concentration at which a gene shows a statistically robust response. This automated POD algorithm is crucial to expand gene expression to interpret dose-response studies that are the foundations of Toxicology and Pharmacology. The POD software tools have been applied to both RNA-seq and RASL-seq data. The Tox21 Genomic Toxicology team has also quantified gene expression responses of endothelial cells, both HUVEC primary cells and endothelial cells derived from iPC stem cells, to 18 tobacco chemicals, using RNA-seq for genome-wide studies.